Oncolytic virus therapy

ABSTRACT

A method of treating a human subject with cancer is disclosed. A pharmaceutical composition is administered to the subject, the pharmaceutical composition comprising human leukocytes and a replication-competent oncolytic virus in suspension in a physiologically acceptable solution. Alternatively the pharmaceutical composition comprises human leukocytes or platelets infected with an oncolytic virus.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/290,051, filed May 11, 2001, the content of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] “Treatment of Neoplasms with Viruses” (WO 00/62735) relates to amethod of administering viruses that are able to replicate and killneoplastic cells with a deficiency in the IFN-mediated antiviralresponse. One specific aspect of this patent involves the “systemic”administration of such viruses.

[0003] Paramyxoviruses are known to interact with erythrocytes andagglutinate them. They are reported to elute from erythrocytes withlower efficiency than influenza viruses. Howe, C. and Lee, L. T. (Adv.Virus Res. 17:1-50, 1972). Furthermore, the specific inhibition ofhemagglutination of erythrocytes caused by paramyxoviruses throughneutralization by antibodies specific to viral coat proteins is a wellunderstood phenomenon.

[0004] Schirrmacher et al (Int. J of Oncology 16:363-373, 2000) haveshown that NDV will activate mouse macrophages for anti-tumor activity.The experimental results appearing below demonstrate that NDV does notpreferentially bind to mouse leukocytes when added to mouse whole blood.

[0005] Bonina et al (Giom. Batt. Virol. Immun., LXXVIII, 254-261, 1985)show that human macrophages could support the growth of NDV.

[0006] Woodruff et al (Cellular Immunology 5:296-306, 1972) and Woodruffand Woodruff (J of Immunology 116(6);2176-2183, 1974) found that NDVagglutinates rat, mouse and human lymphocytes in vitro.

[0007] Faden et al (Blood, 58:221-7,1981) showed that NDV will bind tohuman neutrophils in vitro.

[0008] None of the above indicate any preferential binding of NDV toleukocytes over erythrocytes.

[0009] The literature describes the binding of NDV to red blood cells(usually chicken). As NDV is not pathogenic in man it is a surprisingresult to find that NDV binds preferentially to the white blood cellcomponent of human blood.

[0010] The literature indicates that the binding of NDV to cells occursthrough the interaction of the Neuraminidase of the viral HN proteinwith sialic acid residues attached to cell surface proteins. Humanerythrocytes have a very high density of sialic acid residues attachedto surface proteins. The ratio of erythrocytes to leukocytes in humanblood is approximately 1000 to 1. Thus, it is especially surprising thatNDV binds to the leukocyte fraction instead of the much more numerouserythrocytes.

SUMMARY OF THE INVENTION

[0011] This invention provides a method of treating a human subject withcancer, comprising administering to the subject an amount of apharmaceutical composition effective to treat the subject, thepharmaceutical composition comprising human leukocytes and areplication-competent oncolytic virus in suspension in a physiologicallyacceptable solution, wherein the virus binds specifically to theleukocytes; and the ratio of plaque-forming units of the virus to numberof leukocytes in the composition is at least 1:100, thereby treating thesubject.

[0012] This invention provides a method of treating a human subject withcancer, comprising administering to the subject an amount of apharmaceutical composition effective to treat the subject, thepharmaceutical composition comprising human cells infected with anoncolytic virus in suspension in a physiologically acceptable solution,wherein the cells are leukocytes or platelets, thereby treating thesubject.

[0013] This invention provides the use of a pharmaceutical compositionto treat a human subject with cancer or in the manufacture of amedicament for the treatment of cancer, the pharmaceutical compositioncomprising: (a) human leukocytes and a replication-competent oncolyticvirus in suspension in a physiologically acceptable solution, whereinthe virus binds specifically to the leukocytes and the ratio of plaqueforming units of the virus to number of leukocytes in the composition isat least 1:100; or (b) human cells infected with an oncolytic virus insuspension in a physiologically acceptable solution, wherein the cellsare leukocytes or platelets. Uses (a) and (b) are linked in thatpracticing use (a) generally involves practicing use (b) since thereplication-competent oncolytic virus will generally infect theleukocytes.

[0014] This invention is based, in part, on the finding that anoncolytic virus such as NDV binds to leukocytes and platelets. NDV bindsleukocytes preferentially compared to erythrocytes. Tumors involveinflammatory processes. Therefore leukocytes to which an oncolytic virusis bound or which are infected with an oncolytic virus are aparticularly effective means of delivering oncolytic viruses.

DETAILED DESCRIPTION OF THE INVENTION

[0015] As used herein the term “human cells” means cells isolated from ahuman, or cultured cells that have been derived from cells isolated froma human and/or whose nucleic acid component has been altered by, forexample, immortalization, irradiation or recombinant means. “Humanleukocytes” and “human platelets” are human cells that are leukocytesand platelets, respectively. Except where otherwise specified orrequired by the context the terms “cells” or “human cells” refer toleukocytes and/or platelets.

[0016] As used herein the term “plaque-forming unit” (pfu) means oneinfectious virus particle.

[0017] As used herein the term “multiplicity of infection” (MOI) meansthe number of infectious virus particles added per cell.

[0018] As used herein the term “clonal virus” means a virus derived froma single infectious virus particle and for which individual molecularclones have significant nucleic acid sequence homology. For example, thesequence homology is such that at least eight individual molecularclones from the population of virions have a sequence homology greaterthan 95% over 300 contiguous nucleotides.

[0019] As used herein the term “leukocyte virus complex” (LVC) means thecomplex formed when an oncolytic virus is mixed with a leukocyte cellpopulation and the virus has become associated with the cells. The termincludes both cells where the virus is bound to the outside of the celland cells which are infected by the virus.

[0020] As used herein a virus is said to “bind(s) specifically” to agiven cell if such virus binds to such cell with a greater specificitythan such virus binds to erythrocytes. The terms bind(s) specificallyand specifically bind(s) are used interchangeably.

[0021] As used herein “NDV” is an abbreviation for Newcastle DiseaseVirus.

[0022] As used herein the term “replication-competent” virus refers to avirus that produces infectious progeny in cancer cells.

[0023] As used herein the transitional term “comprising” is open-ended.A claim utilizing this term can contain elements in addition to thoserecited in such claim.

[0024] In accordance with this invention the human cells can be derivedfrom any source. They can be donated by someone other than the subject.However if feasible it is generally preferred to use cells donated bythe subject, for safety reasons. Optionally cells isolated from thedonor can first be grown in culture and the cultured cells are alsoconsidered to be cells of the donor from which they were derived.Examples of cultured cells that can be utilized in accordance with thisinvention include immortalized human leukocyte cell lines. Suitable celllines are publicly available from sources such as the American TypeCulture Collection, for example U-937 (ATCC No. CRL-1593.2) and KG-1(ATCC No. CCL-246).

[0025] The leukocytes utilized in accordance with this invention (e.g.monocytes, neutrophils and lymphocytes including tumor-infiltratinglymphocytes) can be active or inactive. Techniques for inactivatingleukocytes include irradiation.

[0026] The cells utilized in accordance with this invention can beisolated (for example by leukopheresis in the case of leukocytes).However it is not necessary to isolate the cells and whole blood can beused instead, in which case the pharmaceutical composition comprises theoncolytic virus suspended in whole blood or whole blood containingleukocytes and/or platelets infected with the virus. Optionally theleukocytes or platelets are first isolated from whole blood, mixed orinfected with the virus and then added back to the other bloodcomponents.

[0027] In different embodiments of this invention the leukocytes areselected from monocytes, neutrophils and lymphocytes. In a more specificembodiment of this invention the leukocytes are tumor-infiltratinglymphocytes (TILs). TILs may be prepared for example by the methoddescribed in Rabinowich, H., et al., (Cancer Res. 47: 173-7, 1987).

[0028] In accordance with this invention the oncolytic virus utilizedcan be of low (lentogenic), moderate (mesogenic) or high (velogenic)virulence. The level of virulence is determined in accordance with theMean Death Time in Eggs (MDT) test. (Alexander, “Chapter 27: NewcastleDisease” in Laboratory Manual for the Isolation and Identification ofAvian Pathogens, 3^(rd) ed., Purchase, et al. eds. (Kendall/Hunt, Iowa),page 117.) Viruses are classified by the MDT test as lentogenic (MDT>90hours); mesogenic (MDT from 60-90 hours); and velogenic (MDT<60 hours).

[0029] In an embodiment of this invention the virus is a clonal virus.

[0030] Referring to the method or use in which the pharmaceuticalcomposition utilized comprises leukocytes and oncolytic virus insuspension, in an embodiment of such method the ratio of plaque-formingunits of the virus to number of leukocytes in the composition is atleast 1:1. Generally it is preferred that the leukocytes be saturatedwith active virus particles. In the case of NDV saturation is achievedat a 200:1 ratio of plaque-forming units of the virus to number ofleukocytes. Accordingly in an embodiment of this invention the virus isNDV and the ratio of plaque-forming units of the virus to number ofleukocytes in the composition is from about 1:1 to about 200:1, andpreferably is about 200:1.

[0031] In the method or use described above in which the pharmaceuticalcomposition utilized comprises cells infected with an oncolytic virus,in an embodiment of such method the infected cells are at leastone-tenth of one percent (0.1%) of the total number of leukocytes andplatelets in the composition, more preferably at least thirty percentand most preferably about one hundred percent. The virus utilized can bereplication incompetent although preferably it is replication competent.

[0032] In an embodiment of this invention the oncolytic virus isselected from the group consisting of a Newcastle Disease Virus (NDV), aMumps Virus, a Measles Virus, a Vesicular Stomatitis Virus, aPara-influenza Virus, an Influenza Virus, an Adenovirus, a Herpes IVirus, a Vaccinia Virus, and a Reovirus. In a more specific embodiment aNewcastle Disease Virus strain of moderate virulence can be utilized.

[0033] The skilled clinician can determine the optimal amount of thecomposition to be administered in each case. Typically when the cellsare leukocytes the effective amount is a daily dosage of the compositioncontaining from 6×10⁶ to 6×10¹⁰ leukocytes per square meter of patientsurface area, for example about 6×10⁷ leukocytes per square meter ofpatient surface area. When the cells are platelets the effective amountis typically a daily dosage of the composition containing from 10⁹ to10¹¹ platelets per square meter of patient surface area, for exampleabout 10¹¹ platelets per square meter of patient surface area.

[0034] The daily dosage of the composition can be administered to thesubject in multiple administrations in the course of a singletwenty-four hour period in which a portion of the daily dosage isadministered at each administration. More preferably the daily dosage isadministered in a single administration. In an embodiment of thisinvention the daily dosage of the composition is administered to thesubject at a frequency of from one to seven times (i.e. on each of fromone to seven days) in a one-week period.

[0035] In accordance with this invention, any conventional route ofadministration is suitable for administering the pharmaceuticalcomposition. For example the composition can be administeredintravenously, intratumorally, intraperitoneally or intravesicularly(kidneys). In the case of intravenous administration it is convenient ifthe volume of the composition administered is from twenty-fivemilliliters to one liter. In the case of intratumoral administration itis convenient if the volume of composition administered is from onehundred microliters to ten milliliters per tumor mass. In the case ofintraperitoneal administration it is convenient if the volume ofcomposition administered is up to two liters. In the case ofintravesicular administration it is convenient if the volume ofcomposition administered is up to seventy-five milliliters, preferablyfrom fifty to sixty milliliters. Depending on the amount of pfus ofvirus and cells to be administered the concentration of the compositioncan be varied to achieve the desired volume. When the cancer is a solidtumor the composition can be administered by any of the routes givenabove, for example intravenously or intratumorally. When the cancer isother than a solid tumor (e.g. leukemia) the composition is notadministered intratumorally and instead can be administered by the otherroutes given above, for example intravenously.

[0036] The invention will be better understood by reference to thefollowing examples which illustrate but do not limit the inventiondescribed herein. In the following examples the NDV used was atriple-plaque purified attenuated (moderately virulent) version of theMK107 strain of Newcastle Disease Virus, described more fully ininternational patent publication WO 00/62735, published Oct. 26, 2000(Pro-Virus, Inc.).

EXAMPLES Example 1 Binding of NDV to Human Blood Cells

[0037] Purpose:

[0038] Investigate the binding of NDV to human blood cells in order todetermine which cell types bind the virus.

[0039] Materials:

[0040] Reference lot RL-004 NDV; Antibody Mab2F12 (3.5 mg/ml) is raisedto NDV HN protein; BD PharMingen Stain Buffer (PSB) containing 2% fetalbovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10× FACS Lysing Solution, Cat 349202, lot #82026

[0041] Method:

[0042] Human whole blood was collected in citrate tubes (3.2%, 0.105M,Becton Dickinson #366415). Approximately 4 ml from each of 2 tubes waspooled and kept at room temperature until use. Cells were counted usingthe trypan blue exclusion method and a hemocytometer. NDV lot numberRL-004 (1.3E+10 PFU/ml) was used to infect the cells at MOIs(Multiplicity of Infection, expressed as PFU/cell) of 0.2, 0.05 and 0.02(along with a negative control of no added virus). After virus wasadded, tubes were incubated at 37° C. for 30 minutes. Gentle mixing ofthe tubes to keep cells in suspension was performed at 3 intervalsduring the incubation period.

[0043] The samples were washed twice by adding 2 ml of cold PSB,centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solutionaway from the cell pellet. The PSB was removed each time by aspiration.The volumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 g of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described.The goat anti-mouse—PE reporter antibody was added to each sample byadding 1 ml of a 12 μg/ml solution of this antibody. The samples wereagain incubated for 30 minutes on ice and washed as described above. Foranalysis of the leukocyte fraction, 100 μl of each sample was incubatedwith 3 ml of 1× FACS Lysing Solution for 6 minutes at room temperature.The samples were centrifuged and aspirated as before. The cell pelletswere re-suspended in 0.5 ml of PSB. For erythrocyte analysis, 1.5 μl ofeach sample was added to 2.0 ml of PSB. Samples analysis was performedwith a Becton Dickinson FACSCalibur flow cytometer. Forward and sidescatter parameters employed linear settings and FL2 detection ofphycoerythrin employed logarithmic settings.

[0044] Results:

[0045] The results of the experiment shown in Table 1 indicate that NDVbinds preferentially to the leukocyte fraction of human whole blood. Atan MOI of 0.05 (to whole blood), 46% of the leukocytes are positive forNDV compared to 0% of the erythrocytes. At an MOI of 0.2 NDV is presenton 89% of the leukocytes while bound to only 15% of the erythrocytes.Note that MOI's of 0.05 and 0.2 are approximately 5 and 200 to theleukocytes if the presence of the erythrocytes is discounted. The smallamount of NDV binding to erythrocytes at the higher MOI may reflect lowaffinity binding to sialic residues on proteins present on the surfaceof these cells. TABLE 1 % Cells Positive for NDV Binding MOIErythrocytes Leukocytes 0.05 0 46 0.2 15 89

[0046] It is generally appreciated that Paramyxoviridae interact witherythrocytes and produce haemagglutination of the erythrocytes (see How,C. and Lee, L. T., “Virus-Erythrocyte Interactions”). Thus, it iscommonly believed that the major cell type involved in the binding ofthese viruses, including NDV, are the erythrocytes. The binding of NDVto cells is thought to occur through the interaction of theNeuraminidase activity of the viral HN protein to sialic acid residuesattached to cell surface proteins. Human erythrocytes have a very highdensity of sialic acid residues attached to surface proteins for thepurpose of keeping the cells in solution in the blood. As the ratio oferythrocytes to leukocytes in human blood is approximately 1000 to 1, itis especially surprising that NDV binds to the leukocyte fractioninstead of the vastly more numerous erythrocytes.

Example 2 NDV Associates with Leukocytes in the Presence of NDVNeutralizing Antibody

[0047] Introduction/Background:

[0048] The purpose of this experiment was to assess the ability of NDVto bind to leukocytes in the presence or absence of NDV neutralizingantibody. Human clinical patient 521 received approximately 27 treatmentcourses of NDV therapy prior to having whole blood drawn for thisexperiment. Patient 521 displayed significant levels of NDV neutralizingantibodies as detected by plaque neutralization and micro-neutralizationassays.

[0049] Methods:

[0050] Human whole blood from a naive donor and patient 521 werecollected in citrate tubes (3.2%, 0.105M, Becton Dickenson #366415). Thetubes from each donor were kept at room temperature. Whole blood wasdivided into aliquots and spiked with NDV (lot number RL-005). NDV wasspiked at MOIs (multiplicity of infection, expressed as PFU/cell) of0.2, 0.02 and 0.002 (along with a negative control of no added virus).After virus was added, tubes were incubated at room temperature for 30minutes. Plasma and leukocytes were isolated from the spiked samples bygradient centrifugation using polymorphprep (Nycomed, Inc.). Plasma wascarefully removed from the top of the gradient. Two Leukocyte bands (apolymorphonuclear cell band and a mononuclear cell band) were collectedand placed in 0.5× DMEM. Cells were pelleted and resuspended in fullstrength DMEM. Cells were washed several times to remove the separationmedia. Leukocytes in each aliquot were enumerated using a CoulterCounter. Known numbers of leukocytes or volumes of plasma wereco-cultured or inoculated onto monolayers of HT1080 human fibrosarcomacells (ATCC, CCL-121) in 25 cm² tissue culture flasks (Corning). HT1080cells are highly sensitive to cytolysis by NDV. Monolayers wereevaluated qualitatively over several days for the presence of CPE(cytopathic effect). Flasks which exhibited CPE were consideredpositive.

[0051] Results:

[0052] As shown in Table 2, for both the naive donor and patient 521, at300,000 leukocytes per flask for all three MOI spikes, both flaskstested were positive for infectivity by NDV. For the naïve donor, allflasks were positive at the 3000 cell level and all but the lowest MOIspike was positive at the 30 cell level. At the 3000 cell level forpatient 521, 1 of 2 flasks were positive at the 0.02 MOI and 0 of 2 atthe 0.002 MOI. At the 30 cell level for patient 521, 1 of 2 flasks werepositive at the 0.2 MOI, all other flasks at this cell level werenegative.

[0053] The plasma data show that in the naive patient, virus could berecovered from the plasma at all MOIs tested (see Table 3). However, noinfectious virus was recovered from patient 521 plasma which containedneutralizing antibody.

[0054] Discussion:

[0055] The results show that the number of leukocytes associated withinfectious virus was reduced in patient 521 compared to the naïve donor.However, virus was still able to bind to leukocytes in the presence ofneutralizing antibody. This binding was shown to occur even at low MOI(0.002), which is similar to the MOI for patient dosing.

[0056] NDV elicits a humoral immune response which results in theproduction of neutralizing antibody. Binding of virus to leukocytes mayallow the virus to be protected from the neutralizing antibody. This isan advantage over free virus, which is exposed to the neutralizingantibody and rendered non-infectious.

[0057] It can be concluded from these data that using leukocytes as avehicle to deliver virus to tumors is an advantage over free circulatingvirus which would be rendered non-infectious by neutralizing antibody inpatients that have generated an immune response (ie. Patients which havereceived multiple doses of NDV). TABLE 2 Evaluation of Leukocytes forthe Presence of Bound Infectious NDV Number of Positive Cultures AfterLeukocytes are Placed in Co-Culture with a Monolayer of HT1080 NDV SpikeCells in a T25 Flask (MOI) 300,000 Cells 3000 Cells 30 Cells Naïve Donor0.2   2* 2 2 0.02  2 2 2 0.002 2 2 0 No Spike (Ctl.) 0 0 0 Patient 5210.2   2* 2 1 0.02  2 1 0 0.002 2 0 0 No Spike (Ctl.) 0 0 0

[0058] TABLE 3 Evaluation of Plasma for the Presence of Infectious NDVSample Naïve Donor Patient 521 0.2 MOI Spike 1 0 0.02 MOI Spike 1 00.002 MOI Spike 1 0 No Spike (Control) 0 0

[0059] As NDV is not pathogenic in man it is a surprising result to findthat NDV binds preferentially to the white blood cell component of humanblood.

Example 3 Preparation of Human Leukocyte/NDV Complex

[0060] Human Leukocytes (5×10⁺⁸ cells) are prepared by leukophoresis orby gradient centrifugation employing POLYMORPHPREP (Nycomed, Inc.) usingthe manufacturer's instructions. The cells are washed twice in sterile1XPBS at room temperature and brought to a volume of 10 ml in the samebuffer. The cells are mixed with 1×10⁺¹⁰ pfu of NDV (added aseptically)and allowed to sit for 30 minutes (with further brief mixings at 10 and20 minutes). The cells are centrifuged for 5 minutes at 1500 rpm and thePBS removed. The cells are washed with 20 ml of 1XPBS and centrifugedagain. The cell pellet is diluted to 10 ml for injection.

Example 4 Treatment of Human Tumor Xenografts (<10 mm and >5 mm) inAthymic Mice with Human Leukocyte/NDV Complex

[0061] Athymic mice are injected intradermally with 10 million humantumor cells. After tumors reached a size range of between 5 and 10 mm, asingle injection of 8×10⁺⁶ cells of the Leukocyte/NDV complex describedin Example 3 is given. The effect of LVC on tumor growth is examined forcomplete and partial regressions.

Example 5 Systemic Treatment of Human Tumors with Human Leukocyte/NDVComplex in Patients

[0062] Human leukocytes are obtained by leukophoresis from patients. Theleukocyte/NDV complex (LVC) is prepared as described in Example 3. Thecomplex (5×10⁺⁸ cells) is returned to the patient by intravenousinjection. Injections of LVC are given at three-day intervals for 21days.

Example 6 Systemic Treatment of Human Tumors with Human DonorLeukocyte/NDV Complex in Patients

[0063] Human leukocytes are obtained by leukophoresis from naive donors.The leukocyte/NDV complex (LVC) is prepared as described in Example 3.The complex (5×10⁺⁸ cells) is returned to the patient by intravenousinjection. Injections of LVC are given at three-day intervals for 21days.

Example 7 Binding of NDV to Mouse Blood Cells

[0064] Purpose:

[0065] Investigate the binding of NDV to mouse blood cells in order todetermine which cell types bind the virus.

[0066] Materials:

[0067] Reference lot RL-004 NDV; Antibody Mab2F12 (3.5 mg/ml) is raisedto NDV HN protein; BD PharMingen Stain Buffer (PSB) containing 2% fetalbovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10× FACS Lysing Solution, Cat 349202, lot #82026.

[0068] Method:

[0069] Mouse whole blood was collected in citrate tubes (3.2%, 0.105M,Becton Dickinson #366415). Cells were counted using the trypan blueexclusion method and a hemocytometer. NDV lot number RL-005 (4.2E+10PFU/ml) was used to infect the cells at MOIs (Multiplicity of Infection,expressed as PFU/cell) of 0.2, 1 and 3 (along with a negative control ofno added virus). After virus was added, tubes were incubated at 37° C.for 30 minutes. Gentle mixing of the tubes to keep cells in suspensionwas performed at 3 intervals during the incubation period.

[0070] The samples were washed twice by adding 2 ml of cold PSB,centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solutionaway from the cell pellet. The PSB was removed each time by aspiration.The volumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 μg of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described. Agoat anti-mouse—PE reporter antibody was added to each sample by adding1 ml of a 12 μg/ml solution of this antibody. The samples were againincubated for 30 minutes on ice and washed as described. For analysis ofthe leukocyte fraction, 100 μl of each sample was incubated with 3 ml of1× FACS Lysing Solution for 6 minutes at room temperature. The sampleswere centrifuged as before. The cell pellets were re-suspended in 0.5 mlof PSB. For erythrocyte analysis, 1.5 μl of each sample was added to 2.0ml of PSB. Samples analysis was performed with a Becton DickinsonFACSCalibur flow cytometer. Forward and side scatter parameters employedlinear settings and FL2 detection of phycoerythrin employed logarithmicsettings.

[0071] Results:

[0072] The results of the experiment shown in Table 4 indicate thatunlike the binding of NDV to human blood cells where the viruspreferentially binds to leukocytes (Example 1), NDV binds preferentiallyto the erythrocyte fraction of whole mouse blood and does not bind tothe leukocytes. TABLE 4 % Cells Positive for NDV Binding MOIErythrocytes Leukocytes 0.2 30 1 1 80 1 3 92 1

[0073] At an MOI of 0.2 where human leukocytes are mostly positive forthe binding of NDV, mouse leukocytes are negative.

Example 8 Binding of NDV to Rat Blood Cells

[0074] Purpose:

[0075] Investigate the binding of NDV to mouse Rat blood cells in orderto determine which cell types bind the virus.

[0076] Materials:

[0077] Reference lot RL-004 NDV; Antibody Mab2F12 (3.5 mg/ml) is raisedto NDV HN protein; BD PharMingen Stain Buffer (PSB) containing 2% fetalbovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10× FACS Lysing Solution, Cat 349202, lot #82026.

[0078] Method:

[0079] Whole blood (1.8 ml) was collected in citrate tubes (3.2%,0.105M, Becton Dickinson #366415) from a Sprague Dawley rat and kept atroom temperature until use. Cells were counted using the trypan blueexclusion method and a hemocytometer. NDV lot number RL-005 (4.2E+10PFU/ml) was used to infect the cells at MOIs (Multiplicity of Infection,expressed as PFU/cell) of 0.05, 0.2, 0.5, 1 and 3 (along with a negativecontrol of no added virus). After virus was added, tubes were incubatedat 37° C. for 30 minutes. Gentle mixing of the tubes to keep cells insuspension was performed at 3 intervals during the incubation period.

[0080] The samples were washed twice by adding 2 ml of cold PSB,centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solutionaway from the cell pellet. The PSB was removed each time by aspiration.The volumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 μg of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described. Agoat anti-mouse—PE reporter antibody was added to each sample by adding1 ml of a 12 μg/ml solution of this antibody. The samples were againincubated for 30 minutes on ice and washed as described. For analysis ofthe leukocyte fraction, 100 μl of each sample was incubated with 3 ml of1× FACS Lysing Solution for 6 minutes at room temperature. The sampleswere centrifuged as before. The cell pellets were re-suspended in 0.5 mlof PSB. For erythrocyte analysis, 1.5 μl of each sample was added to 2.0ml of PSB. Samples analysis was performed with a Becton DickinsonFACSCalibur flow cytometer. Forward and side scatter parameters employedlinear settings and FL2 detection of phycoerythrin employed logarithmicsettings.

[0081] Results:

[0082] The results of the experiment shown in Table 5 indicate that NDVbinds to rat leukocytes at a low MOI (0.02 and 0.05) while it does notbind well to the erythrocytes at these MOI's. The pattern appears to beintermediate between the binding of the virus to human leukocytes towhich it binds preferentially and to mouse leukocytes, to which it doesnot bind. TABLE 5 % Cells Positive for NDV Binding MOI ErythrocytesLeukocytes 0.02  3  7 0.05 12 21 0.2 30 29 1 63 58 3 90 66

Example 9 Binding of NDV to Human Platelets

[0083] Purpose:

[0084] Investigate the binding of NDV to human platelets

[0085] Materials:

[0086] Reference lot RL-004 NDV; Antibody Mab2F12 (3.5 mg/ml) is raisedto NDV HN protein; BD PharMingen Stain Buffer (PSB) containing 2% fetalbovine serum and 0.02% sodium azide, catalog #554656, lot M059394;Rockland goat anti-mouse polyclonal antibody—phycoerythrin conjugate,catalog #710-1832, lot 7367; Becton Dickinson Immunocytometry Systems10× FACS Lysing Solution, Cat 349202, lot #82026.

[0087] Method:

[0088] Isolation of Platelets

[0089] Whole human blood was collected in citrate tubes (3.2% 0.105MCitrate, Becton Dickinson #366415). Seven milliliters of whole bloodwere placed into a 15 ml polypropylene centrifuge tube. The tube wascentrifuged at 800 g for 5 minutes at room temperature. Approximately1.5 ml of platelet-rich plasma (PRP) was collected from the top of thecentrifuge tube. Based on published values for yield, the sample wasdetermined to contain 8.0E+8 cells/ml. 100% of PRP was infected with NDV(RL-005) at MOIs of 0.1, 10 and 100 for 30 minutes at room temperature.

[0090] The samples were washed twice by adding 2 ml of cold PSB,centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solutionaway from the cell pellet. The PSB was removed each time by aspiration.The volumes of each sample were adjusted to 1 ml by adding cold PSB. Themonoclonal antibody Mab2F12 was added to each sample by adding 20 ul ofa solution containing 9.1 μg of the antibody. The samples were incubatedfor 30 minutes on ice and washed twice again as previously described. Agoat anti-mouse—PE reporter antibody was added to each sample by adding0.1 ml of a 12 μg/ml solution of this antibody. The samples were againincubated for 30 minutes on ice and washed as described. Sample analysiswas performed with a Becton Dickinson FACSCalibur flow cytometer.Forward and side scatter parameters employed linear settings and FL2detection of phycoerythrin employed logarithmic settings.

[0091] Results:

[0092] The results of the experiment shown in Table 6 indicate that NDVbinds to human platelets. The number of platelets that are positive forbinding of NDV does not increase greatly in the MOI range testedalthough the mean fluorescence does, indicating that more NDV binds tothe positive portion of the platelets as the MOI increases. TABLE 6 NDVBinding To Human Platelets % Positive Mean MOI Platelets Fluorescence 00 10 1 67 258 10 81 616 100 73 1005

Example 10 Binding Hierarchy of NDV to Human Leukocytes

[0093] Materials:

[0094] Reference lot RL-004; Antibody Mab2F12 (3.5 mg/ml) was raised toNDV HN protein; BD PharmMingen™ Stain Buffer (PSB) containing 2% fetalbovine serum and 0.02% sodium azide, catalog # 544656, lot M059394;Rockland goat anti-mouse polyclonal antibody-phycoerythrin conjugate,catalog #7100-1832, lot 7367; Becton Dickinson Immumocytometry Systems™10× FACS Lysing Solution Cat. 349202, Lot #82026.

[0095] Method:

[0096] Human whole blood was collected in citrate tubes (3.2%, 0.105M,Becton Dickinson™ #366415). Approximately 4 ml from each of two tubeswas pooled and kept at room temperature until use. Cells were countedusing the trypan blue exclusion method and a hemocytometer. NDV lotnumber RL-004 (1.3E+10 PFU/ml) was used to infect 100 μl samples ofwhole blood at MOIs (Multiplicity of Infection, expressed as PFU/cell)of 0, 0.005, 0.01, 0.02, 0.05, 0.1, and 0.2 to the whole blood. Aftervirus was added, samples were incubated at 37° C. for 30 minutes.

[0097] The samples were washed twice by adding 2 ml of cold PSB,centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solutionaway from the pellet. The volumes of each sample were adjusted to 100μlwith PSB. Monoclonal antibody Mab2F12 was added to each sample by adding2 μl of a solution made in PSB containing 1 μg of the antibody. Thesamples were incubated for 30 minutes on ice and washed twice again aspreviously described. The goat anti-mouse—PE reporter antibody was addedto each sample by adding 100 μl of a 12 μg/ml solution of this antibodydiluted 2.4:1 in PSB. The samples were incubated for 30 minutes on iceand washed as described above. To prepare the samples for leukocyte cellanalysis, each sample was incubated with 3 ml of 1× FACS Lysing Solutionfor 6 minutes at room temperature, then centrifuged and aspirated asbefore. The cell pellets were re-suspended in 0.5 ml of PSB. Sampleanalysis was performed with a Becton Dickinson FACSCalibur™ flowcytometer. Granulocyte, lymphocyte and monocyte populations were gatedby comparing the forward scatter and side scatter parameters for eachsample. This was used to determine the number of cells positive forbinding the virus and the mean fluorescence value of each of thesepopulations for each sample.

[0098] The results (Table 7) indicate that NDV binds preferentially tothe three cell populations. The order of preference is:monocyte>granulocyte>>lymphocyte. Furthermore, as shown from the meanfluorescence data, the monocyte population binds substantially more NDVthan does the granulocyte population, and binds much more virus thandoes the lymphocyte cell population. TABLE 7 % Positive Cells MeanFluorescence MOI Monocyte Granulocyte Lymphocyte Monocyte GranulocyteLymphocyte 0 2 2 2 13 19 7 0.005 80 68 19 65 20 9 0.01 92 97 39 134 3511 0.02 99 100 54 219 66 16 0.05 98 100 72 314 113 22 0.10 99 100 84 448154 31 0.20 100 100 96 692 251 69

What is claimed is:
 1. A method of treating a human subject with cancer,comprising administering to the subject an amount of a pharmaceuticalcomposition effective to treat the subject, the pharmaceuticalcomposition comprising human leukocytes and a replication-competentoncolytic virus in suspension in a physiologically acceptable solution,wherein the virus binds specifically to the leukocytes; and the ratio ofplaque-forming units of the virus to number of leukocytes in thecomposition is at least 1:100, thereby treating the subject.
 2. Themethod of claim 1, wherein the leukocytes are leukocytes of the subject.3. The method of claim 1, wherein the leukocytes are leukocytes of adonor other than the subject.
 4. The method of claim 1, wherein theleukocytes are active.
 5. The method of claim 1, wherein the leukocytesare inactive.
 6. The method of claim 1, wherein the leukocytes arecultured.
 7. The method of claim 6, wherein the cultured leukocytescomprise an immortalized cell line.
 8. The method of claim 7, whereinthe immortalized cell line is U-937 or KG-1.
 9. The method of claim 1,wherein the leukocytes are leukocytes isolated by leukopheresis.
 10. Themethod of claim 1, wherein the pharmaceutical composition comprises theoncolytic virus suspended in whole blood.
 11. The method of claim 1,wherein the leukocytes are monocytes.
 12. The method of claim 1, whereinthe leukocytes are neutrophils.
 13. The method of claim 1, wherein theleukocytes are lymphocytes.
 14. The method of claim 13, wherein thelymphocytes are tumor-infiltrating lymphocytes.
 15. The method of claim1, wherein the virus is of low to moderate virulence.
 16. The method ofclaim 1, wherein the virus is a clonal virus.
 17. The method of claim 1,wherein the ratio of plaque-forming units of the virus to number ofleukocytes in the composition is at least 1:1.
 18. The method of claim1, wherein the virus is selected from the group consisting of aNewcastle Disease Virus, a Mumps Virus, a Measles Virus, a VesicularStomatitis Virus, a Para-influenza Virus, an Influenza Virus, anAdenovirus, a Herpes I Virus, a Vaccinia Virus, and a Reovirus.
 19. Themethod of claim 18, wherein the virus is a Newcastle Disease Virus. 20.The method of claim 19, wherein the virus is a Newcastle Disease Virusof moderate virulence.
 21. The method of claim 18, wherein the ratio ofplaque-forming units of the virus to number of leukocytes in thecomposition is from about 1:1 to about 200:1.
 22. The method of claim21, wherein the ratio is about 200:1.
 23. The method of claim 1, whereinthe effective amount is a daily dosage containing from 6×10 ⁶ to 6×10¹⁰leukocytes per square meter of patient surface area.
 24. The method ofclaim 23, wherein the daily dosage contains about 6×10⁷ leukocytes persquare meter of patient surface area.
 25. The method of claim 23,wherein the daily dosage is administered in single administration. 26.The method of claim 23, wherein the daily dosage is administered at afrequency of from one to seven times in a one-week period.
 27. Themethod of claim 1, wherein the composition is administeredintravenously.
 28. The method of claim 27, wherein the volume of thecomposition administered is from twenty-five milliliters to one liter.29. The method of claim 1, wherein the composition is administeredintratumorally.
 30. The method of claim 29, wherein the volume ofcomposition administered is from one hundred microliters to tenmilliliters per tumor mass.
 31. The method of claim 1, wherein thecomposition is administered intraperitoneally.
 32. The method of claim31, wherein the volume of composition administered is up to two liters.33. The method of claim 1, wherein the composition is administeredintravesicularly.
 34. The method of claim 33, wherein the volume ofcomposition administered is up to seventy-five milliliters.
 35. Themethod of claim 34, wherein the volume of composition administered isfrom fifty to sixty milliliters.
 36. The method of claim 1, wherein thecancer is a solid tumor and the composition is administeredintravenously or intratumorally.
 37. The method of claim 1, wherein thecancer is other than a solid tumor and the composition is administeredintravenously.
 38. The method of claim 37, wherein the cancer isleukemia.
 39. A method of treating a human subject with cancer,comprising administering to the subject an amount of a pharmaceuticalcomposition effective to treat the subject, the pharmaceuticalcomposition comprising human cells infected with an oncolytic virus insuspension in a physiologically acceptable solution, wherein the cellsare leukocytes or platelets, thereby treating the subject.
 40. Themethod of claim 39, wherein the cells are cells of the subject.
 41. Themethod of claim 39, wherein the cells are cells of a donor other thanthe subject.
 42. The method of claim 39, wherein the cells are cultured.43. The method of claim 42, wherein the cultured cells comprise animmortalized cell line.
 44. The method of claim 43, wherein theimmortalized cell line is U-937 or KG-1.
 45. The method of claim 39,wherein the cells are leukocytes.
 46. The method of claim 43, whereinthe leukocytes are active.
 47. The method of claim 43, wherein theleukocytes are inactive.
 48. The method of claim 39, wherein theleukocytes are monocytes.
 49. The method of claim 39, wherein theleukocytes are neutrophils.
 50. The method of claim 39, wherein theleukocytes are lymphocytes.
 51. The method of claim 50, wherein thelymphocytes are tumor-infiltrating lymphocytes.
 52. The method of claim39, wherein the leukocytes are leukocytes isolated by leukopheresis. 53.The method of claim 39, wherein the cells are platelets.
 54. The methodof claim 39, wherein the pharmaceutical composition comprises wholeblood containing leukocytes or platelets infected with the virus. 55.The method of claim 39, wherein the virus is of low to moderatevirulence.
 56. The method of claim 39, wherein the virus is a clonalvirus.
 57. The method of claim 39, wherein the infected cells are atleast one-tenth of one percent of the total number of leukocytes andplatelets in the composition.
 58. The method of claim 57, wherein theinfected cells are at least thirty percent of the total number ofleukocytes and platelets in the composition.
 59. The method of claim 39,wherein the virus is selected from the group consisting of a NewcastleDisease Virus, a Mumps Virus, a Measles Virus, a Vesicular StomatitisVirus, a Para-influenza Virus, an Influenza Virus, an Adenovirus, aHerpes I Virus, a Vaccinia Virus, and a Reovirus.
 60. The method ofclaim 59, wherein the virus is a Newcastle Disease Virus.
 61. The methodof claim 60, wherein the virus is a Newcastle Disease Virus of moderatevirulence.
 62. The method of claim 39, wherein the effective amount is adaily dosage containing from 6×10⁶ to 6×10¹⁰ leukocytes per square meterof patient surface area.
 63. The method of claim 62, wherein the dailydosage contains about 6×10⁷ leukocytes per square meter of patientsurface area.
 64. The method of claim 62, wherein the daily dosage isadministered in a single administration.
 65. The method of claim 62,wherein the daily dosage is administered at a frequency of from one toseven times in a one-week period.
 66. The method of claim 39, whereinthe effective amount is a daily dosage containing from 10⁹ to 10¹¹platelets per square meter of patient surface area.
 67. The method ofclaim 66, wherein the daily dosage contains about 10¹¹ platelets persquare meter of patient surface area.
 68. The method of claim 66,wherein the daily dosage is administered in a single administration. 69.The method of claim 66, wherein the daily dosage is administered at afrequency of from one to seven times in a one-week period.
 70. Themethod of claim 39, wherein the composition is administeredintravenously.
 71. The method of claim 70, wherein the volume of thecomposition administered is from twenty-five milliliters to one liter.72. The method of claim 39, wherein the composition is administeredintratumorally.
 73. The method of claim 72, wherein the volume ofcomposition administered is from one hundred microliters to tenmilliliters per tumor mass.
 74. The method of claim 39, wherein thecancer is a solid tumor and the composition is administeredintravenously or intratumorally.
 75. The method of claim 39, wherein thecancer is other than a solid tumor and the composition is administeredintravenously.
 76. The method of claim 75, wherein the cancer isleukemia.
 77. The method of claim 39, wherein the composition isadministered intraperitoneally.
 78. The method of claim 77, wherein thevolume of composition administered is up to two liters.
 79. The methodof claim 78, wherein the composition is administered intravesicularly.80. The method of claim 79, wherein the volume of compositionadministered is up to seventy-five milliliters.
 81. The method of claim80, wherein the volume of composition administered is from fifty tosixty milliliters.
 82. The method of claim 39, wherein the virus isreplication competent.